The potential use of ribozymes as pharmaceuticals is an exciting prospect. An important requirement for the development of ribozyme pharmaceutical products is the ability to specifically target a ribozyme to a cellular RNA of interest. This can be especially difficult when designing a ribozyme for a chimeric RNA molecule that is homologous to a second RNA molecule, particularly if cleavage of the second RNA is detrimental to the host. It may be equally difficult to target an RNA molecule that is folded in a way that prevents ribozyme interactions at or near the ribozyme cleavage site. We have encountered both of these problems in our attempts to design ribozymes that are specific for an aberrant mRNA associated with chronic myelogenous leukemia (CML).
Chronic Myelogenous Leukemia (CML) and acute lymphocytic leukemia (ALL) represent two different types of leukemias. CML is a chronic myeloproliferation disorder associated with the cytogenic marker called the Philadelphia chromosome (Nowell, P. C. and Hungerford, D. A., Science 1960, 132, 1497) in approximately 95% of patients. The Philadelphia chromosome is a chromosomal abnormality resulting from reciprocal translocations between chromosomes 9 and 22 (Mercola, M. et al., Science 1933, 221, 663).
The breakpoints on chromosome 22 are clustered in a 6 Kb region termed the breakpoint cluster region (bcr) (Groffen, J. et al., Cell 1984, 36, 93-99), while on chromosome 9, the breakpoints are scattered throughout a 90 Kb region upstream from c-abl exon 2 (Heisterkamp, N. et al., Nature 1983, 306, 239-242). The resultant fusion transcripts, which are about 8.5 kb long, contain bcr sequences upstream and abl sequences downstream.
The cellular gene abl, a highly conserved gene, represents the progenitor of the viral transforming gene (v-abl) of Abelson leukemia virus. v-abl confers to Abelson leukemia virus the ability to transform a broad range of hematopoietic cell types. Transformation is mediated by a tyrosine kinase encoded by the viral genome, composed of v-abl polypeptide attached at its N-terminus to viral gag polypeptide. The human abl gene was mapped to chromosome 9, and is expressed as a 145 kd protein having tyrosine kinase activity. Misregulation of abl is implicated in CML in humans. Shtivelman et al., Cell 1986, 47, 277-284.
The various 9:22 translocations associated with the Philadelphia chromosome can be subdivided into two types: K28 translocations and L6 translocations. In the K28 mRNA, abl exon 2 is linked to bcr exon 3. In the L-6 mRNA, abl exon 2 is linked to bcr exon 2. C-myc mRNA is pertinent for blast crisis in CML.
A third type of 9:22 translocation has also been identified. The chromosome 9 breakpoints specific for this type of translocation are located 5' of the L6 breakpoints. The presence of this abnormal chromosome, however, is associated with the establishment of acute lymphocytic leukemia (ALL) not CML. Selleri et al., Blood 1990, 75, 1146-1153. Further, the K28 and L6 mRNAs have been associated with some patients with ALL.
Much emphasis has been placed on the role of mRNA K28 and the establishment of CML. (Shtivelman, E. et al., Cell 1986, 47, 277-284; Kubonishi, I. and Miyoshi, I., Int. J. Cell Cloning 1983, 1, 105-117; Shtalrid, M. T. et al., Blood 1988, 72, 485-490; Mills, K. I. et al., Blood 1988, 72, 1237-1241).
In the K28 translocations, the chromosomal 22 breakpoints lie between bcr exons 3 and 4 (Shtivelman, E. et al., Cell 1986, 47, 277-284). Transcription through this region yields an mRNA which can be alternatively spliced to yield two distinct mRNAs (Shtivelman, E. et al., Cell 1986, 47, 277-284): mRNA K28 and mRNA L6. In mRNA K28, bcr exon 3 is fused to abl exon 2, while in mRNA L6, bcr exon 2 is fused to abl exon 2. Importantly, the mRNA yielded can change during the course of disease. In the L6 translocations, the chromosomal breakpoints lie between bcr exons 2 and 3 (Shtivelman, E. et al., Cell 1986, 47, 277-284). Transcription through this region yields only one species of mRNA, mRNA L6. The K28 and the L6 mRNAs encode a protein with an aberrant tyrosine kinase activity which is unique to CML cells and which is believed to play a key role in the establishment of CML. McLaughlin et al., Proc. Nat'l. Acad. Sci. 1987, 84, 6558-6562.
To date, bone marrow transplantation has been the most effective way to treat CML. Nonetheless, using a more sensitive technique, it has been demonstrated that in some patients receiving ablative radiation and/or chemotherapy followed by bone marrow transplantation, residual leukemia cells may persist. Researchers have detected residual bcr-abl mRNA in patients following bone marrow transplantation in a study using a more sensitive PCR assay. Snyder et al., Transplantation 1991, 51, 1033-1040. There remains an unmet need in reducing the level of mRNAs and their protein products implicated in leukemias in the treatment of CML and ALL. These mRNAs include the bcr-abl transcripts such as the K28 and L6 mRNAs, as well as c-myc mRNA.
Ribozymes offer an attractive alternative. Chimeric RNAs, such as those occurring in CML, can be ideal candidates for ribozyme targeting particularly when a ribozyme cleavage site is located within 2 or 3 nucleotides of the chimeric junction, i.e. near the junction. In this case a ribozyme can be targeted specifically to the chimeric molecule but not the non-chimeric molecule by specifying that 1) ribozyme sequences 5' of the catalytic region be complementary to chimeric RNA sequences located immediately 3' of the cleavage site, and 2) ribozyme sequences 3' of the catalytic region be complementary to chimeric sequences immediately 5' of the cleavage site. The specificity of the ribozyme is thus, presumably, maintained and the potentially harmful results of non-specific ribozyme cleavage can be avoided.
However, not all chimeric mRNAs exhibit a convenient site for ribozyme cleavage so near to the junction site. Examination of the L6 bcr-abl mRNA sequence reveals that the closest "NUX" ribozyme cleavage sites in the vicinity of the bcr-abl junction are located 7, 8, and 19 nucleotides away from the junction (see FIG. 1b). It is not feasible to target any of these sites for ribozyme cleavage in the manner described because such ribozymes would likely also cleave normal abl mRNA or normal bcr mRNA. In addition, computer predictions for the secondary structures of L6 bcr-abl mRNA suggest that these sites may be inaccessible to conventional ribozymes. Accordingly, we initiated new approaches to the design of ribozymes specific for L6 bcr-abl mRNA.
Reddy, et al., WO 92/00080, published Jan. 9, 1992, report a ribozyme capable of cleaving the hybrid bcr-abl "gene" of CML at or near the breakpoint. The translocation product targeted was not specified, and the implication was that only one translocation occurred. Nevertheless, from sequence complementarity, it appears that the ribozyme of Reddy was directed against the K28 translocation. This translocation exhibits a convenient site near the junction for conventional ribozyme cleavage. Tests verifying specificity of the ribozyme for hybrid bcr-abl were not provided.
As noted previously, the particular message transcribed by K28 can change during the course of disease. Further, certain CML patients express the L6 mRNA either alone or in addition to the K28 mRNA. Shtivelman et al., Cell 1986, 47, 277-284. There remains an unsolved problem which has not been addressed; the L6 mRNA. The L6 mRNA will most likely be unaffected by treatments designed solely to target the K28 mRNA. Further, there is an unmet need in targeting the c-myc mRNA in conjunction with the bcr-abl transcripts.
The present invention provides oligonucleotide therapeutics and methods of treating CML and ALL whereby the aforementioned transcripts are specifically targeted. The present invention also provides ribozymes capable of cleaving L6 mRNA, or both L6 and K28 mRNA.
The present invention also addresses, in general, an unmet need for ribozymes capable of cleaving a target mRNA in which the catalytic recognition sequence is located at a distance from the nucleation site, the junction of a chimeric target mRNA, or in which catalytic recognition sequences are not readily accessible due to secondary structure. The ribozymes of the present invention are useful, for example, in the treatment of diseases involving translocations, such as CML, ALL and follicular lymphoma.